**2.** *In vitro* **propagation**

Sugarcane micropropagation enables the identical production of the selected cultivars using PTC techniques. PTC refers to growing and differentiation of cells, tissues and organs isolated from the mother plant, on artificial semisolid or liquid media under aseptic and controlled conditions. The small organs or pieces of tissue used in PTC are called explants. PTC medium provides inorganic nutrients and usually a carbohydrate to replace the carbon which the plant normally fixes from the atmosphere by photosynthesis. When carbon is supplied with sucrose and kept in low light conditions, micropropagated plantlets are not fully dependent on their own photosynthesis.

To date, sugarcane micropropagation has shown great productive potential [3]; it is being used in commercial laboratories to obtain certified plant material.

The sugarcane micropropagation process is carried out in the following stages [9].

by sowing bud-containing cuttings. However, this technique does not ensure the sanitation and rejuvenation of the selected varieties in the field. An alternative to this problem is the use of Plant Tissue Culture (PTC) techniques [4]; this plant biotechnology tool allows the establishment, manipulation and development, under artificial and controlled conditions, of cells, tissues or organs and is very useful for the regeneration of rejuvenated, genetically homoge-

In sugarcane, *in vitro* propagation or cloning of plants uses PTC techniques to obtain a constant supply of plant material, unlike conventional vegetative propagation, which is seasonal in nature. Sugarcane micropropagation has allowed the rapid multiplication of new varieties, rejuvenation of old deteriorated varieties and sanitation of diseased varieties [3] and has also facilitated the exchange of in vitro plant material. Currently, semiautomation of micropropagation by temporary immersion systems (TISs) offers a practical strategy to reduce production costs [5]. TISs are semiautomated bioreactors designed for the mass propagation of cells, tissues, embryos or organs using liquid medium [6]. TISs have been shown to be a powerful

A micropropagation-based crop has prominently better quality than a conventionally raised one. According to Sawant et al. [1], sugarcane micropropagation increases productivity in the field by up to 25%, while Pérez et al. [8] mention that the combined effect of in vitro sanitation and rejuvenation is expected to increase sugar yields by between 10 and 15% per unit area. These advantages have allowed the commercial exploitation of micropropagation in the sugar industry worldwide. This technology is now used to supplement commercial sugarcane production in many countries including Brazil, India, the USA and

Certified vitroplants obtained from in vitro propagation systems are used in the field for the production of quality seed. This technology helps farmers to enhance crop productivity. The aim of this chapter is to report, from the laboratory to the field, a seed production system comprising basic seed (primary seed), foundation (secondary seed) and commercial seed pro-

Sugarcane micropropagation enables the identical production of the selected cultivars using PTC techniques. PTC refers to growing and differentiation of cells, tissues and organs isolated from the mother plant, on artificial semisolid or liquid media under aseptic and controlled conditions. The small organs or pieces of tissue used in PTC are called explants. PTC medium provides inorganic nutrients and usually a carbohydrate to replace the carbon which the plant normally fixes from the atmosphere by photosynthesis. When carbon is supplied with sucrose and kept in low light conditions, micropropagated plantlets are not fully dependent on their

To date, sugarcane micropropagation has shown great productive potential [3]; it is being

used in commercial laboratories to obtain certified plant material.

neous plants free of pests and diseases.

102 Sugarcane - Technology and Research

tool for sugarcane propagation [7].

duction to obtain a commercial crop.

**2.** *In vitro* **propagation**

own photosynthesis.

Cuba [4].

**Stage 0: mother plant selection.** Donor cultivars are selected and conditioned to be used to initiate in vitro cultures. It should be considered that the mother plant corresponds to the selected variety; in many cases, there is a varietal mixture in commercial cane plantations. The genetic purity of the variety should be certified by the breeder or research organization identified for the maintenance of the variety.

**Stage I: in vitro establishing.** The choice of the apical meristems (explants) and their disinfection is carried out to initiate an aseptic in vitro culture. Apical meristem culture produces virus-free sugarcane plants. The meristem remains in an active state during the vegetative growth phase, and the meristem cells are in a permanent totipotent state.

**Stage II: multiplication.** It is at this stage that mass propagation is performed, obtaining many new shoots from minimal amounts of tissue. Based on our experience, we recommend making no more than eight subcultures because above that level the length and number of shoots decrease. In addition, the likelihood of genetic variants occurring increases.

**Stage III: elongation and rooting.** The shoots must form their root system and at the same time increase their size to facilitate their manipulation and adaptation to the acclimatization conditions.

**Stage IV: acclimatization.** This process is carried out in a greenhouse. It consists of a slow reduction of the relative humidity and gradual increases in the luminous intensity for a better adaptation to the external environment. The greenhouse infrastructure must ensure control of both relative humidity and light entry.

It is important to mention that the elongation and rooting stage varies according to the method being utilized; it is not always necessary when semiautomation of micropropagation by TISs is used.

Conventional micropropagation of sugarcane in semisolid media has been reported [10, 11]. However, to reduce the labor required and increase efficiency, temporary immersion systems (TISs) have been successfully used to improve in vitro sugarcane multiplication [12–16]. The principle of these systems is the immersion of explants for a determined time and frequency.

We have implemented different TISs for commercial sugarcane micropropagation. Sugarcane meristems (cv. Mex 69–290) were collected from field-grown plants and cultured following the protocol of Jiménez et al. [17] The 3-cm-long sugarcane shoots after three subcultures (30 d each) were used as explant. Explants (two shoots each) were placed in the Temporary Immersion Bioreactor (TIB™, Cuba), the Recipient for Automated Temporary Immersion (RITA™, France), the Gravity Immersion Bioreactor (GIB, Mexico) and the SETIS™ Bioreactor (Belgium) containing MS [18] medium supplemented with 30 g/L sucrose, 1 mg/L Kinetin (Sigma Chemical Company, MO, USA), 0.6 mg/L 3-indoleacetic acid (IAA, Sigma Chemical Company, MO, USA) and 0.3 mg/L 6-benzylaminopurine (BAP, Sigma Chemical Company, MO, USA). The pH of the culture medium was adjusted to 5.8 with 0.1 N sodium hydroxide and then autoclaved at 1.2 kg/cm<sup>2</sup> for 15 min at 120°C. Three replicates were used in all experiments. TISs were incubated at 24 ± 2°C and were maintained under fluorescent light (40–50 μmol m−2 s−1) and a photoperiod of 16 h. Immersion frequency was according to Lorenzo et al. [12] After 30 d of incubation, the number and length of shoots per explant were assessed.

A completely randomized experimental design was used for all experiments. Results were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey's comparison of means test (*p* ≤ 0.05) using SPSS statistical software (version 22 for Windows).

**3. Genetic homogeneity**

with respect to the mother plant.

subcultures the length and number of shoots decrease.

be micropropagated.

percentage of subcultures 1–10.

The genetic or epigenetic variation obtained by different in vitro propagation systems is called somaclonal variation [19]; it is a problem that affects commercial micropropagation, where it is necessary to maintain the maximum genetic homogeneity of the regenerated individuals

*In Vitro* Propagation of Sugarcane for Certified Seed Production

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The causes of somaclonal variation are not well understood and have not been fully elucidated [20]. However, some factors that determine the frequency of somaclonal variation include the in vitro regeneration system, the type and concentration of growth regulators applied, and the number of subcultures [21]. Consequently, it is important to determine the optimal number of subcultures that can be made from an explant for each sugar cultivar to

Martínez-Estrada et al. [22] determined by inter-simple sequence repeat (ISSR) markers that no more than eight subcultures should be done due to the existence of polymorphism between the subcultures produced by a Temporary Immersion Bioreactor (**Figure 2**), since above eight

Genetic homogeneity and plant health are two important quality aspects that must be addressed before the seedlings are distributed outside the laboratory. According to Lal et al. [3], contamination of cultures is a severe problem that not only reduces the frequency of shoot culture initiation from the source explants but also the total number of shoots produced at various cycles of cultures. Plant tissues could also be cultured in the presence of bacterial and/

**Figure 2.** Effect of subculturing on polymorphism percentage of shoots of sugarcane (cv. Mex 69–290) using Temporary Immersion Bioreactors assessed by inter-simple sequence repeat (ISSR) markers. Each bar represents the polymorphic

or fungal contaminants. Therefore, a phytosanitary diagnosis should be required.

When evaluating the different TISs in sugarcane during in vitro propagation, significant statistical differences were observed for the number and length of shoots per explant. The bioreactors with the highest number of shoots per explant were TIB, GIB and SETIS, with 38, 40 and 41 shoots/explant, respectively, followed by RITA, with 32 shoots/explant. Regarding shoot length, the bioreactors with the longest length were TIB, GIB and SETIS with 8.6, 10.7 and 9.8 cm in length, followed by RITA with 6.0 cm in length (**Table 1**).

Semiautomation of sugarcane micropropagation using TISs is a strategy to reduce production costs. The TIB, GIB and SETIS bioreactors showed good performance in the formation of the length and number of shoots; probably their size, among other factors, favors the development of explants. On the other hand, RITA, due to its limited capacity, did not allow an increase in length and number of new shoots. Commercial sugarcane micropropagation by TISs is shown in **Figure 1**.


Values represent mean ± SE (standard error). Means with different letters per column represent statistical difference (Tukey, *p* ≤ 0.05).

**Table 1.** Sugarcane (*Saccharum* spp. hybrid cv. Mex 69-290 micropropagation by different temporary immersion systems (TISs)).

**Figure 1.** Sugarcane micropropagation by temporary immersion systems. (a) Recipient for automated temporary immersion (RITA™), (b) gravity immersion bioreactor (GIB), (c) Temporary Immersion Bioreactor (TIB) and (d) SETIS™ bioreactor, after 30 d of incubation.
